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The necessity to understand the influence of global ocean change on biota has exposed wide-ranging gaps in our knowledge of the fundamental principles that underpin marine life. Concurrently, physiological research has stagnated, in part driven by the advent and rapid evolution of molecular biological techniques, such that they now influence all lines of enquiry in biological oceanography. This dominance has led to an implicit assumption that physiology is outmoded, and advocacy that ecological and biogeochemical models can be directly informed by omics. However, the main modeling currencies are biological rates and biogeochemical fluxes. Here, we ask: how do we translate the wealth of information on physiological potential from omics-based studies to quantifiable physiological rates and, ultimately, to biogeochemical fluxes? Based on the trajectory of the state-of-the-art in biomedical sciences, along with case-studies from ocean sciences, we conclude that it is unlikely that omics can provide such rates in the coming decade. Thus, while physiological rates will continue to be central to providing projections of global change biology, we must revisit the metrics we rely upon. We advocate for the co-design of a new generation of rate measurements that better link the benefits of omics and physiology.

Si cycling is linked with processes from global carbon sequestration to community composition and is especially important in aquatic ecosystems. Lake Michigan has seen dramatic fluctuations in dissolved silica (dSi) over several decades, which have been examined in the context of planktonic processes (diatom blooms), but the role of benthic organisms (macroalgae and their epiphytes) in Si cycling have not been explored. To assess significance of nearshore benthic algae in Si dynamics, we assembled dSi data from an offshore site sampled since the late 1980’s, and sampled off three Milwaukee beaches during 2005–19. Using colorimetric assays and alkaline digestion, we measured dSi, biogenic silica in particulate suspended material (pSi) and biogenic silica in benthic macroalgae (Cladophora) and epiphytic diatoms (bSi). Offshore, dSi increased about 1 μM per year from 25 μM in the late 1980’s to nearly 40 μM in 2019. Nearshore dSi fluctuated dramatically annually, from near zero to concentrations similar to offshore. Both Cladophora and its epiphytes contained significant bSi, reaching up to 30% of dry mass (300 mg Si g dry mass-1) of the assemblage in summer. Microscopic analyses including localization with a Si-specific-stain and X-ray microanalysis showed bSi in epiphytic diatom cells walls, but the nature and localization of Si in macroalgae remained unclear. A simple model was developed estimating Si demand of algae using the areal macroalgal biomass, growth rates inferred from P-content, and bSi content, and comparing Si demand with dSi available in the water column. This indicated that 7–70% of the dSi in water overlying nearshore benthic algal beds could be removed per day. Key elements of the Si cycle, including which organisms sequester bSi and how rapidly Si is recycled, remain unclear. This work has implications for coastal marine waters where large macroalgal biomass accumulates but bSi content is virtually unknown.

Understanding and predicting changes in phytoplankton populations requires knowledge of losses due not only to sedimentation and grazing, but also to intrinsic processes (here, collectively termed ���cell death���). Cell death is poorly understood, especially in freshwater phytoplankton, but experiments in culture often suggest involvement of abiotic factors (e.g. temperature, light, nutrients). The occurrence of cell death was examined in a simple, natural environment: a small, well-mixed, temperate, urban pond during a period of phytoplankton growth, from mid-July to mid-November. Abundances of 18 phytoplankton taxa were measured weekly and fluorescence microscopy and staining was used to detect dead cells (using SYTOX which measures loss of membrane integrity) and cells undergoing cell death (using Annexin-V, which measures lipid inversions of membranes, an early signal of cell death). Dead and dying cells occurred in most phytoplankton taxa, but incidence and timing varied considerably, e.g. species like the chlorophyte Ankistrodesmus spiralis showed 20���30% of cells staining with SYTOX and Annexin in late autumn when the population was decreasing, while the dinoflagellate Peridinium sp. showed staining of up to 50% of cells with STYOX throughout the period, and the cyanobacterium Microcystis aeruginosa occasionally showed staining of 100% of cells with SYTOX. Overall, there was some association between cell death staining and growth phase with 10���15% of the total community showing SYTOX and Annexin staining in late autumn, when most populations were declining. Cell death could not be correlated with thresholds or rapid changes in abiotic conditions (e.g. temperature, irradiance) or with indicators of nutrient limitation (e.g. N:P ratios). While abiotic factors have been clearly implicated in cell death within unialgal culture experiments, in natural freshwater ecosystems interactions between biotic factors, such as pathogens or allelopathy, may play greater roles in losses related to cell death and be distinct for different taxa.

Metacaspases and paracaspases are proteases that were first identified as containing a caspase-like structural fold (Uren et al., 2000). Like caspases, metacaspases and paracaspases are multifunctional proteins regulating diverse biological phenomena, such as aging, immunity, proteostasis, and programmed cell death. The broad phylogenetic distribution of metacaspases and paracaspases across all kingdoms of life and large variation of their biochemical and structural features complicate classification and annotation of the rapidly growing number of identified homologs. Establishment of an adequate classification and unified nomenclature of metacaspases and paracaspases is especially important to avoid frequent confusion of these proteases with caspases—a tenacious misnomer that unfortunately does not appear to decline with time. This Letter represents a consensus opinion of researchers studying different aspects of caspases, metacaspases, and paracaspases in various organisms, ranging from microbes to plants and animals., This work was supported by the Knut and Alice Wallenberg Foundation.

Integration of algae and cyanobacteria with microbial fuel cell bioelectrochemical systems (BES) can significantly improve energy recovery and nutrient remediation in wastewater treatment. One innovative option is an integrated photobioelectrochemical system (IPB). Algae can contribute to BES function as an organic feedstock to support bacterial growth, by assisting anode bacteria to generate electricity, by providing oxygen from photosynthesis as a cathode electron acceptor, and by removing N and P from effluent water. However, critical interactions among bacteria-algae communities are poorly understood and practical questions such as light and pH conditions and taxa selection need more research to optimize microbial interactions and promote IPB function. Only a few ‘lab weed’ algal and cyanobacterial taxa have been tried in IPB systems but algae offer additional metabolic flexibility such as mixotrophy, to further process organic carbon, and nutrient hyperaccumulation, which have yet to be examined for potential in wastewater IPB treatment systems. This review aims to serve as a guide for wastewater bioenergy engineers to address challenges in IPB systems, and identifies a need for more collaboration between algal biologists and engineers to optimize algal-microbial community collaboration and work towards improved sustainability of wastewater treatment.

The critical roles of viruses in nutrient cycling are well established for marine ecosystems, but less is known for freshwater viruses about diversity, seasonality, and relationships with productivity and ecosystem trophic status. This study aimed to examine virus morphotypes and abundance in relation to prokaryotic cell abundance and chlorophyll a concentrations in three seasonal temperate freshwater sites representing eutrophic, mesotrophic, and oligotrophic status in Lake Michigan watershed. We found strong correlations between virus-like particle abundance (VLPA) and prokaryote abundance (PA) and chlorophyll concentration across the trophic gradient (r = 0.744, P

High pressure liquid chromatography (HPLC) has become a standard for analysis of phytoplankton pigment in marine and freshwaters. In marine systems, such data have been combined with optimization algorithms (e.g., CHEMTAX) to quantitatively predict the taxa present in a given sample; such studies are less common in freshwaters, particularly in oligo- and mesotrophic lakes. HPLC/CHEMTAX methods were compared with taxonomic identification and quantification using traditional microscopy and volume to chlorophyll a conversions in Lake Michigan phytoplankton communities collected on six cruises during summer (June to August) 2008. Chlorophyll a reached maxima (approximately 1.5 μg L − 1 nearshore and 0.5 μg L − 1 offshore) in late June/early July, with the exception of the offshore metalimnion where chlorophyll a peaked (3.0 μg L − 1 ) in early July. Taxonomic groups were consistently misidentified by HPLC/CHEMTAX, relative to microscope methods; of 18 points of comparisons, only 5 were significantly related (judged by regression of relative proportion of chlorophyll a estimated by HPLC/CHEMTAX versus cell counts/volume). Confusion between diatoms and chrysophytes was particularly serious. For example, in late July when counts indicated that biovolume at epilimnetic stations was dominated by diatoms, HPLC/CHEMTAX indicated dominance by chrysophytes. Grouping diatoms and chrysophytes as a single taxon improved comparisons, so that there was significant agreement in 7 out of 18 cases. While some specific improvement to the HPLC method (e.g., selecting a smaller subset of pigments relevant to the specific phytoplankton assemblages found) might help, our work emphasizes the point made repeatedly in the literature, that HPLC methods cannot replace microscopy.

Several fish species, including the walleye (Sander vitreus), have “yellow” and “blue” color morphs. In S. vitreus, one source of the blue color has been identified as a bili-binding protein pigment (sandercyanin), found in surface mucus of the fish. Little is known about the production of the pigment or about its functions. We examined the anatomical localization and seasonal variation of sandercyanin in S. vitreus from a population in McKim Lake, northwestern Ontario, Canada. Skin sections were collected from 20 fish and examined histologically. Mucus was collected from 306 fish over 6 years, and the amount of sandercyanin was quantified spectrophotometrically. Sandercyanin was found solely on dorsal surfaces of the fish and was localized to novel cells in the epidermis, similar in appearance to secretory sacciform cells. Sandercyanin concentrations were significantly higher in fish collected in summer versus other seasons. Yellow and blue morphs did not differ in amounts of sandercyanin, suggesting that the observed blue color, in fact, arises from lack of yellow pigmentation in blue morphs. The function of the sandercyanin remains unclear, but roles in photoprotection and countershading are consistent with available data.

Over recent decades, Lake Michigan phytoplankton and zooplankton communities have experienced dramatic changes driven both from the bottom (e.g. P loading reduction) and top (e.g. predatory cladoceran invasions) of the food web. We used two data sets from nearly identical sampling at an offshore station (100 m depth) in western Lake Michigan (1988–92 and 2007–09), to test for bottom-up effects (i.e. declines in chlorophyll a (chl a ) or increases in particulate C:P, leading to declines in P-rich cladocerans versus copepods), and top-down effects of invasive predatory cladocerans (i.e. declines in native zooplankton from predation or competition). Between the two periods, total P and particulate C declined, while nitrate and silicate increased. While chl a in the largest cells (> 53 μm and 10–53 μm fractions) decreased, particulate C:P ratios were unchanged. Total zooplankton abundance and biomass declined significantly between sampling periods, notably cyclopoid copepods, but not Bosmina or Daphnia species, nor the invasive cladoceran, Bythotrephes longimanus . Bottom-up effects, usually associated with ‘benthification’ attributed to Dreissena grazing, are more consistent with the changes observed than are effects of invasive predatory cladocerans. Differences in observations from those in eastern Lake Michigan or other lake-wide surveys are difficult to reconcile but seem more likely due to temporal differences in sampling rather than spatial ones. Discerning the trajectory of Lake Michigan will require better accounting for zooplankton life histories, more sophisticated understanding of nutritional quality and diet for zooplankton, and clearer coupling of pelagic–benthic cycles of elements, including Si and N.

Viruses are ubiquitous in aquatic ecosystems and play a critical role in nutrient cycling because viral lysis of cells releases phosphorus, an essential and often limiting nutrient. Previous models have examined dynamics of bacterial and viral communities, but with limited analysis and without explicit consideration of nutrients. A recent model ( Fuhrman et al., 2011 ; Math. Comp. Model. 53, 716-730) incorporated internal nutrient content of bacteria and viruses. In the present study, we modified and tested the model with data from natural planktonic communities from Lake Michigan. Replicate 20 L water samples (135 μm screened), were either untreated or enriched with 8 μM phosphate, and bacterial and viral abundance, chl a fluorescence, and phosphorus (total and dissolved inorganic) were monitored for two weeks. Fuhrman et al.'s model (modified to include phytoplankton) was applied to the data, fitting burst size, lytic latent period and virus decay rate. For enriched samples, model fits were good and parameters were consistent with measurements in other freshwater ecosystems. However, for unenriched samples, where nutrient concentrations approached detection limits, model fits were relatively poorer. The model predicted similar viral decay rates but higher burst sizes and longer latent period in phosphorus-limited versus enriched conditions, underlining the potential importance of nutrients in host–virus interactions. The model is likely to be most useful in meso- to eutrophic systems; requirements for future model development and parameter estimation for application to oligotrophic lakes are discussed.

Despite a well-documented rise in nitrate concentration over the past century, Lake Superior has retained an oligotrophic character. In part, this status results from physical attributes of the lake including low temperatures and prolonged isothermy, resulting in deep-mixing and light limitation which constrain primary production. Lake Superior is also phosphorus deficient which limits phytoplankton growth. We conducted large (20 l) volume factorial bioassay experiments to assess the influence of light and nutrients (P, Fe) on nitrate assimilation by a Lake Superior chlorophyte alga. Bioassays seeded with the chlorophyte yielded a strong response to light result- ing in the rapid depletion of nitrate. High light resulted in higher activities of the key N-assimilation enzyme nitrate reductase (NR) and increased algal biomass compared to low light treatments. NR activity was highly correlated with rates of nitrate incorporation in bioassays and field surveys suggesting that NR occupies a critical place in nitrate metabolism. In bioassays, the addition of nutrients (P, Fe) only slightly increased the rate at which nitrate became depleted. Parallel trials using a luminescent cyano- bacterial bioreporter confirmed the lack of response by added nutrients supporting light as an important factor in constraining nitrate assimilation by phytoplankton in the lake.

There can be significant intraspecific individual-level heterogeneity in the intracellular P of phytoplankton, which can affect the population-level growth rate. Several mechanisms can create this heterogeneity, including phenotypic variability in various physiological functions (e.g., nutrient uptake rate). Here, we use modeling to explore the contribution of various mechanisms to the heterogeneity in phytoplankton grown in a laboratory culture. An agent-based model simulates individual cells and their intracellular P. Heterogeneity is introduced by randomizing parameters (e.g., maximum uptake rate) of daughter cells at division. The model was calibrated to observations of the P quota of individual cells of the centric diatom Thalassiosira pseudonana , which were obtained using synchrotron X-ray fluorescence (SXRF). A number of simulations, with individual mechanisms of heterogeneity turned off, then were performed. Comparison of the coefficient of variation (CV) of these and the baseline simulation (i.e., all mechanisms turned on) provides an estimate of the relative contribution of these mechanisms. The results show that the mechanism with the largest contribution to variability is the parameter characterizing the maximum intracellular P, which, when removed, results in a CV of 0.21 compared to a CV of 0.37 with all mechanisms turned on. This suggests that nutrient/element storage capabilities/mechanisms are important determinants of intrapopulation heterogeneity.

Fishes in aquaria and aquaculture settings may experience a variety of stressors including crowding, different lighting, periods of food deprivation, and vibrations from sources including pumps and tapping of tank sides. The effects of such low-level chronic stress are poorly explored. We used replicate sets of six Zebrafish Danio rerio in four series of experiments to compare the effects of (1) stocking densities ranging from 0.13 to 1.2 fish/L, (2) cool white (6,500 K), warm white (4,100 K), and ultraviolet-enhanced (420 actinic) fluorescent lighting, (3) food deprivation for up to 9 d, and (4) random mechanical tapping on the tank side sufficient to induce a startle response on specific behaviors (fin display, body fluttering, aggression, mouth gaping, and chattering), dissolved cortisol released into aquarium water (collected on a chromatography column and analyzed with an immunoassay), and heat-shock proteins (HSPs 27, 40, 60, and 70) detected immunochemically in western blots of muscle tissue. Of all the treatments, only food deprivation resulted in significant differences between control and treatment fish; dissolved cortisol declined after 120 h of starvation and HSP40 and HSP60 in muscle tissue increased significantly after 216 h. High variability in behaviors and HSP measurements was noted within all controls and treatments, suggesting that effects of treatments were experienced unequally by individuals within a treatment. Social stressors resulting from dominance hierarchies may play a critical role in modifying the effects of aquarium and aquaculture stressors on captive fish.

When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of programmed cell death with many similarities to apoptosis is induced, including the induction of caspase-like proteases. Many uncertainties about the regulation and mediators that participate in the process remain. To examine the relationship between caspase-like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX-green staining, and the generation of reactive oxygen species (ROS), we used the broad-range caspase inhibitor Boc-D-FMK to block the activity of the whole class of caspase-like proteins simultaneously. In the presence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase-like enzymes in an alga. The fact that caspase-like inhibitors prevent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase-like proteases might have regulatory roles early in cell death, in addition to dismantling functions.

Measurements of enzymes that mediate uptake and transformations of nutrients are important tools for aquatic microbial ecologists in understanding biogeochemical cycling. Nitrate reductase (NR) functions at a central regulatory step in nitrogen assimilation and so is a critical target for assessing nitrogen availability and use. Importantly, recent work demonstrates that NR can provide such information even when nitrogen is not the primary limiting resource, and also provides data about organismal genetic and functional diversity, and even insight into stress metabolism. NR measurements offer a means to explore complex interactions involving symbioses and cell–cell communication and also help integrate genomic, transcriptomic, and proteomics approaches to understanding microbial ecology and ecosystem dynamics. Moreover, NR has practical applications including biosensor and bioremediation technologies.

Chlorophyll a fluorescence of microalgae is a compelling indicator of toxicity of dissolved water contaminants, because it is easily measured and responds rapidly. While different chl a fluorescence parameters have been examined, most studies have focused on single species and/or a narrow range of toxins. We assessed the utility of one chl a fluorescence parameter, the maximum quantum yield of PSII (F(v)/F(m)), for detecting effects of nine environmental pollutants from a range of toxin classes on 5 commonly found freshwater algal species, as well as the USEPA model species, Pseudokirchneriella subcapitata. F(v)/F(m) declined rapidly over

In this work we develop and analyze a mathematical model describing the dynamics of infection by a virus of a host population in a freshwater environment. Our model, which consists of a system of nonlinear ordinary differential equations, includes an intrinsic quota, that is, we use a nutrient (e.g., phosphorus) as a limiting element for the host and potentially for the virus. Motivation for such a model arises from studies that raise the possibility that on the one hand, viruses may be limited by phosphorus (Bratbak et al. [17]), and on the other, that they may have a role in stimulating the host to acquire the nutrient (Wilson [18]). We perform an in-depth mathematical analysis of the system including the existence and uniqueness of solutions, equilibria, asymptotic, and persistence analysis. We compare the model with experimental data, and find that biologically meaningful parameter values provide a good fit. We conclude that the mathematical model supports the hypothesized role of stored nutrient regulating the dynamics, and that the coexistence of virus and host is the natural state of the system.

Measurements of the efficiency of photosystem (PS) II have become widespread in bio- logical oceanography, and various forms are used to assess the 'health' of marine phytoplankton and to help estimate primary productivity. Absolute values of PS II efficiency depend to some extent on the measuring system, but changes in PS II efficiency are most commonly interpreted in terms of cel- lular acclimations to changing irradiance (including photoacclimation and photoinhibition) and nutri- ent availability (especially N and Fe). Recent measurements of phytoplankton viability in the surface ocean have revealed that in many regions phytoplankton assemblages may contain large proportions of dead cells. The effect of these dead cells on apparent PS II efficiency is largely unknown. By mix- ing live and dead cells and measuring PS II efficiency, we show that the presence of photosyntheti- cally non-functional (dead) cells has surprisingly little effect; in a number of species, mixtures in which 50% of the cells were dead had values of 0.5, similar to values often found in natural assem- blages. A simple model indicates that the non-linear nature of the fluorescence ratio is responsible for this unexpected result. We conclude that relatively high values of PS II efficiency cannot be used as evidence of low mortality. Our findings highlight the need for more information on the physiological status of both eukaryotic and prokaryotic microalgae in nature.

Intertidal macroalgae Fucus and Laminaria experience seasonally fluctuating inorganic N supply. This study examined the effects of long-term N deprivation, recovery following N resupply, and effects of elevated ammonium and nitrate exposure on N acquisition in intertidal algae using manipulations of N supply in tank culture. Over 15 weeks of N deprivation, internal N and nitrate reductase activity (NRA) declined, but maximum quantum yield of PSII was unaffected in Fucus serratus and Fucus vesiculosus. Low NRA was maintained despite no external nitrate availability and depletion of internal pools, suggesting a constitutive NRA, insensitive to N supply. Nitrate resupplied to N-starved thalli was rapidly taken up and internal nitrate pools and NRA increased. Exposure to elevated (50 microM) nitrate over 4 days stimulated nitrate uptake and NRA in Laminaria digitata and F. serratus. Exposure to elevated ammonium suppressed NRA in L. digitata but not in F. serratus. This novel insensitivity of NRA to ammonium in Fucus contrasts with regulation of NRA in other algae and higher plants. Ammonium suppression of NRA in L. digitata was not via inhibition of nitrate uptake and was independent of nitrate availability. L. digitata showed a higher capacity for internal nitrate storage when exposed to elevated ambient nitrate, but NRA was lower than in Fucus. All species maintained nitrate assimilation capacity in excess of nitrate uptake capacity. N uptake and storage strategies of these intertidal macroalgae are adaptive to life in fluctuating N supply, and distinct regulation of N metabolism in Fucus vs Laminaria may relate to position in the intertidal zone.

Fucus and Laminaria species, dominant seaweeds in the intertidal and subtidal zones of the temperate North Atlantic, experience tidal cycles that are not synchronized with light:dark (L:D) cycles. To investigate how nutrient assimilation is affected by light cycles, the activity of nitrate reductase (NR) was examined in thalli incubated in outdoor tanks with flowing seawater and natural L:D cycles. NR activity in Laminaria digitata (Huds.) Lamour. showed strong diel patterns with low activities in darkness and peak activities near midday. This diel pattern was controlled by light but not by a circadian rhythm. In contrast, there was no diel variation in NR activity in Fucus serratus L., F. vesiculosus (L.) Lamour., and F. spiralis L. either collected directly from the shore or maintained in the outdoor tanks. In laboratory cultures, transfer to continuous darkness suppressed NR activity in L. digitata, but not in F. vesiculosus; continuous light increased NR activity in L. digitata but decreased activity in F. vesiculosus. Furthermore, 4 d enrichment with ammonium (50 μmol · L⁻¹ pulses), resulted in NR activity declining by >80% in L. digitata, but no significant changes in F. serratus. Seasonal differences in maximum NR activity were present in both genera with activities highest in late winter and lowest in summer. This is the first report of NR activity in any alga that is not strongly regulated by light and ammonium. Because light and tidal emersion do not always coincide, Fucus species may have lost the regulation of NR by light that has been observed in other algae and higher plants.

Several marine diatoms produce polyunsaturated aldehydes (PUAs) that have been shown to be toxic to a wide variety of model organisms, from bacteria to invertebrates. However, very little information is available on their effect on phytoplankton. Here, we expand previous studies to six species of marine phytoplankton, belonging to different taxonomic groups that are well represented in marine plankton. The effect of three PUAs, 2 E ,4 E -decadienal, 2 E ,4 E -octadienal and 2 E ,4 E -heptadienal, was assessed on growth, cell membrane permeability, flow cytometric properties and morphology. A concentration-dependent reduction in the growth rate was observed for all cultures exposed to PUAs with longer-chained aldehydes having stronger effects on growth than shorter-chained aldehydes. Clear differences were observed among the different species. The prymnesiophyte Isochrysis galbana was the most sensitive species to PUA exposure with a lower threshold for an observed effect triggered by mean concentrations of 0.10 μmol L −1 for 2 E ,4 E -decadienal, 1.86 μmol L −1 for 2 E ,4 E -octadienal and 3.06 μmol L −1 for 2 E ,4 E -heptadienal, and a 50% growth inhibition (EC 50 ) with respect to the control at 0.99, 2.25 and 5.90 μmol L −1 for the three PUAs, respectively. Alternatively, the chlorophyte Tetraselmis suecica and the diatom Skeletonema marinoi (formerly S. costatum ) were the most resistant species with 50% growth inhibition occurring at concentrations at least two to three times higher than I. galbana . In all species, the three PUAs caused changes in flow cytometric measures of cell size and cell granulosity and increased membrane permeability, assessed using the viability stain SYTOX Green. For example, after 48 h 51.6 ± 2.6% of I. galbana cells and 15.0 ± 1.8% of S. marinoi cells were not viable. Chromatin fragmentation was observed in the dinoflagellate Amphidinium carterae while clear DNA degradation was observed in the chlorophyte Dunaliella tertiolecta . Concentrations used are in a significant range for affecting growth and performance of phytoplankton living in close vicinity of PUA-producing algae. Thus, PUAs may act as allelochemicals by mediating interactions among planktonic organisms.

Diatoms are unicellular brown algae that likely arose from the endocytobiosis of a red alga into a single-celled heterotroph and that constitute an algal class of major importance in phytoplankton communities around the globe. The first whole-genome sequence from a diatom species, Thalassiosira pseudonana Hasle et Heimdal, was recently reported, and features that are central to diatom physiology and ecology, such as silicon and nitrogen metabolism, iron uptake, and carbon concentration mechanisms, were described. Following this initial study, the basic cellular systems controlling cell signaling, gene expression, cytoskeletal structures, and response to stress have been cataloged in an attempt to obtain a global view of the molecular foundations that sustain such an ecologically successful group of organisms. Comparative analysis with several microbial, plant, and metazoan complete genome sequences allowed the identification of putative membrane receptors, signaling proteins, and other components of central interest to diatom ecophysiology and evolution. Thalassiosira pseudonana likely perceives light through a novel phytochrome and several cryptochrome photoreceptors; it may lack the conserved RHO small-GTPase subfamily of cell-polarity regulators, despite undergoing polarized cell-wall synthesis; and it possesses an unusually large number of heat-shock transcription factors, which may indicate the central importance of transcriptional responses to environmental stress. The availability of the complete gene repertoire will permit a detailed biochemical and genetic analysis of how diatoms prosper in aquatic environments and will contribute to the understanding of eukaryotic evolution.

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO(3) (-) storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO(3) (-), with highest values in winter, when ambient NO(3) (-) was maximum, and declined with NO(3) (-) during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 +/- 33 nmol NO(3) (-) min(-1) g(-1) in F. vesiculosus versus 55 +/- 17 nmol NO(3) (-) min(-1) g(-1) in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO(3) (-) assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7-50.0, yet seaweeds stored significant NO(3) (-) (up to 40-86 micromol g(-1)). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.

Cell death is a fundamental process in all metazoan organisms. In contrast, the ecological role of cell death in phytoplankton has been sorely neglected: the causes and biochemistry of cell death, and the quantitative significance of cell death in the ecology of phytoplankton populations and in broader biogeochemical cycles, are not well understood. Metazoan cell death is much better described, due to its accepted roles in the regulation of multicellular life. In metazoan cells, an influential paradigm suggests that there are two morphological outcomes of cell death, one caused by an ‘active’ pathway within the cell (so-called ‘programmed cell death’; PCD), and the other from a ‘passive’ externally-driven process (necrosis). Here, we examine the development of this paradigm, and associated concepts, in plant, animal, and microbial life, and discuss the role of cell death amongst the diverse taxa of the phytoplankton. Several recent studies suggest PCD operates in cyanobacteria, chlorophytes, and dinoflage...

When the chlorophyte alga Dunaliella tertiolecta is placed in darkness, a form of programmed cell death with many similarities to apoptosis (including the induction of caspase-like proteases) is induced. Many uncertainties about this process remain, two of which are whether it requires protein synthesis and whether there is potential viral involvement. In order to examine the relationship between the induction and/or activation of proteolytic activities and the cell death event, we used inhibitors of cytoplasmic protein synthesis (cycloheximide, CHX), organellar protein synthesis (chloramphenicol, CAP) and DNA synthesis (mitomycin C, MMC). Use of MMC also allowed us to examine whether temperate viruses were present in the D. tertiolecta isolate, since MMC treatment has been shown to induce lytic cycles. Addition of protein synthesis inhibitors (100 mM CHX or 1500 mM CAP applied singly or both inhibitors added together) did not prevent cell death from occurring when cultures were placed in the dark. There were no differences in caseinolytic activities visualized using zymograms, or in caspase 1, 3, 8 and 9 -like activities. Surprisingly, 100 mM MMC prevented the cell death event. Caseinolytic activities that appeared in darkness in controls did not appear in MMC-treated cultures, and caspase-like activities remained the same as in controls maintained in the light. The lack of effect of CHX and CAP suggests that the cell death programme we observe does not depend on protein synthesis, but rather on post-translational modification of preexisting proteins. Results for MMC discount the involvement of temperate virus, but are difficult to interpret. MMC affects DNA synthesis and presumably transcription, but since inhibitors of translation did not prevent cell death, it is not clear why inhibiting transcription would. MMC affects cell cycle progression and cell division cycles, thus these processes may play as yet unexplained roles in mediating the cell death process observed.

Macroalgal epiphytes within seagrass meadows make a significant contribution to total primary production by assimilating water column N and transferring organic N to sediments. Assim- ilation of NO3 - requires nitrate reductase (NR, EC 1.6.6.1); NR activity represents the capacity for NO3 - assimilation. An optimised in vitro assay for determining NR activity in algal extracts was applied to a wide range of macroalgae and detected NR activity in all 22 species tested with activity 2 to 290 nmol NO3 - min -1 g -1 frozen thallus. With liquid-N2 freezing immediately after sample collec- tion, this method was practical for estimating NR activity in field samples. Vertical distribution of NR activity in macroalgal epiphytes was compared in contrasting Posidonia sinuosa and Amphibolis antarctica seagrass meadows. Epiphytes on P. sinuosa had higher mass-specific NR activity than those on A. antarctica. In P. sinuosa canopies, NR activity increased with distance from the sediment surface and was negatively correlated with (NH4 + ) in the water but uncorrelated with (NO3 - ). This supported the hypothesis that NH4 + released from the sediment suppresses NR in epiphytic algae. In contrast, the vertical variation in NR activity in macroalgae on A. antarctica was not statistically sig- nificant although there was a weak correlation with (NO3 - ), which increased with distance from the sediment. Estimated capacities for NO3 - assimilation in macroalgae epiphytic on seagrasses during summer (24 and 46 mmol N m -2 d -1 for P. sinuosa and A. antarctica, respectively) were more than twice the estimated N assimilation rates in similar seagrasses. When the estimates were based on annual average epiphyte loads for seagrass meadows in other locations, they were comparable to those of seagrasses. We conclude that epiphytic algae represent a potentially important sink for water-column nitrate within seagrass meadows.

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p

Maerl is a general term used for loose-lying subtidal beds of nodular coralline red algae. Maerl beds support high associated invertebrate and algal biodiversity, and are subject to European and UK conservation legislation. Previous investigations have shown European maerl to be ecologically fragile due to growth rates of approximately 1 mm per year. However, these very slow growth rates have hampered attempts to determine the key ecological requirements and sensitivity characteristics of living maerl. In this study, photosynthetic capacity determined by pulse amplitude modulated (PAM) fluorometry was used as a diagnostic of stress caused by various environmental conditions. Maerl species were exposed to a range of temperatures, salinities and light levels and to burial, fragmentation, desiccation and heavy metal treatment. Maerl was not as susceptible as previously assumed to extremes of salinity, temperature and heavy metal pollution, but burial, especially in fine or anoxic sediments, was lethal or caused significant stress. These data indicate that the main anthropogenic hazard for live maerl and the rich communities that depend on them is smothering by fine sediment, such as that produced by trawling or maerl extraction, from sewage discharges or shellfish and fish farm waste, and sedimentation resulting from disruption to tidal flow. 2004 Elsevier Ltd. All rights reserved.

The study of cell death in higher plants and animals has revealed the existence of an active ('programmed') process in most types of cell, and similarities in cell death between plants, animals, yeast and bacteria suggest an evolutionarily ancient origin of programmed cell death (PCD). Despite their global importance in primary production, information on algal cell death is limited. Algal cell death could have similarities with metazoan cell death. One morphotype of metazoan PCD, apoptosis, can be induced by light deprivation in the unicellular chlorophyte Dunaliella tertiolecta. The situation in other algal taxa is less clear. We used a model dinoflagellate (Amphidinium carterae) to test whether mortality during darkness and culture senescence showed apoptotic characteristics. Using transmission electron microscopy, fluorescent biomarkers, chlorophyll fluorescence and particulate carbon analysis we analysed the process of cell mortality and found that light deprivation caused mass mortality. By contrast, fewer dead cells (5-20% of the population) were found in late-phase cultures, while a similar degenerate cell morphology (shrunken, chlorotic) was observed. On morphological grounds, our observations suggest that the apoptotic cell death described in D. tertiolecta does not occur in A. carterae. Greater similarity was found with paraptosis, a recently proposed alternative morphotype of PCD. A paraptotic conclusion is supported by inconclusive DNA fragmentation results. We emphasize the care that must be taken in transferring fundamental paradigms between phylogenetically diverse cell types and we argue for a greater consistency in the burden of proof needed to assign causality to cell death processes.

Understanding the dynamics of upwelling systems, especially the interactions between nutrients and light, has benefited from the application of models of varying complexity. Validation of such models using unialgal cultures or field observations has often proven difficult, but short-term incubations of contained natural assemblages and use of instantaneous physiological indicators offer an alternative approach. In May and June 1996, phytoplankton communities deep in the euphotic zone were sampled from nearly identical physical environments. Replicate samples (20 l volume) were incubated on deck at 50% surface irradiance with either no nutrient additions (Controls) or additions of 20 µM nitrate (Enrichments). Over 24 h, variable fluorescence (Fv:Fm), nitrate reductase activity (NR), nutrients, chlorophyll a and particulate C and N were monitored. Initial chlorophyll a (∼3 µ gl −1 ), phosphate (∼0.2 µM), nitrate (∼1.5 µM) and silicate (∼3 µM) were similar in both months. Changes in NR and Fv:Fm indicated clear physiological responses to changes in irradiance and added nitrate that differed between months. In May, Controls and Enrichments responded in the same way. Fv:Fm stayed constant (0.5), chlorophyll a increased slightly, and NR activity increased markedly in all samples. In contrast, in June, treatments responded quite differently. Fv:Fm was near the theoretical maximum (0.7–0.8) initially and remained constant in Enrichments, but fell sharply in Controls. Declines in controls were also seen for chlorophyll a, and NR activity. Thus, the addition of 20 µM nitrate had a significant effect even though ambient levels of nitrate (>1 µM) should not have been limiting. Small (

Apoptosis is essential for normal growth and development of multicellular organisms, including metazoans and higher plants. Although cell death processes have been reported in unicellular organisms, key elements of apoptotic pathways have not been identified. Here, we show that when placed in darkness, the unicellular chlorophyte alga Dunaliella tertiolecta undergoes a form of cell death reminiscent of apoptosis in metazoans. Many morphological criteria of apoptotic cell death were met, including an increase in chromatin margination, degradation of the nucleus, and DNA fragmentation. Biochemical assays of the activities of cell death-associated proteases, caspases, measured using highly specific fluorogenic substrates, increased with time in darkness and paralleled the morphological changes. The caspase-like activities were inhibited by caspase-specific inhibitors. Antibodies raised against mammalian caspases cross-reacted with specific proteins in the alga. The pattern of expression of these immunologically reactive proteins was correlated with the onset of cell death. The occurrence of key components of apoptosis, and particularly a caspase-mediated cell death cascade in a relatively ancient linage of eukaryotic photoautotrophs, argues against current theories that cell death evolved in multicellular organisms. We hypothesize that key elements of cell death pathways were transferred to the nuclear genome of early eukaryotes through ancient viral infections in the Precambrian Ocean before the evolution of multicellular organisms and were subsequently appropriated in both metazoan and higher plant lineages.

Proteases are essential components of cells and participate in processes ranging from photoacclimation and nutrient acquisition to development and stress responses. Virtually nothing is known about this diverse group of enzymes in macroalgae. Methods to measure proteases developed for phytoplankton (caseinolysis, leucine aminopeptidase (LAP) activity and casein zymograms) have been modified to allow use with spectrophotometers as well as spectrofluorometers, so making them accessible to a wider number of users. Applying the methods to a wide range of macroalgae from intertidal zones in south-western Spain and Northern Ireland detected proteases in all species examined. Protease activities differed among species but there was little systematic variation with taxonomic group. Moreover, similar species in Spain and Northern Ireland often showed differences. With the exception of LAP activities in brown algae, protease activities were one to two orders of magnitude greater in macroalgae than those previously measured in phytoplankton, when expressed per unit protein. However, scaling of activities to protein was complicated by significant interferences. Copper-based protein assays gave erroneously high results when applied to the brown algae, and partial purification of proteins by trichloroacetic acid precipitation did not overcome the problem. The higher levels of protease activity suggest that proteolysis plays a more significant role in multicellular than in unicellular algae. Proteases were characterized in terms of activities at different pH and the effect of protease inhibitors. In common with microalgae but in contrast to findings for animals and higher plants, proteases tended to show higher activities in the neutral and alkaline ranges. Many commonly used protease inhibitors (e.g. leupeptin and phenylmethylsulphonyl fluoride) had relatively little effect, which may explain why biochemical work with macroalgal species has traditionally been difficult. Our results suggest that macroalgal proteases are easily measurable but highly variable. A major source of variability that has not been assessed is differing environmental conditions. If this is correct, measurements of proteolytic enzymes may provide a valuable tool for examining biologically relevant changes in environments. Controlled laboratory experiments and seasonal monitoring are the next logical steps towards this goal.

Although temperature effects on phytoplankton growth and photosynthesis can be clearly demonstrated in the laboratory, their relevance in the field is much harder to establish. Recently, however, it has been recognized that temperature has a significant influence on nitrogen uptake. In particular, temperate marine diatom species may be limited by their ability to acquire nitrate at temperatures above approximately 16°C. In order to explore this idea, we grew the diatom Thalassiosira pseudonana at 8, 17 and 25°C, and compared cell composition, and rates of growth (µ), 15 N incorporation, calculated nitrate incorporation (the product of µ and cell N content), and the activ- ity of nitrate reductase (NR), a key enzyme involved in nitrate incorporation. Cell N content, protein and volume remained relatively constant across different temperatures, but cell C, chlorophyll a (chl a), and C:N ratio increased with increasing temperature, suggesting that C metabolism was affected more strongly than N metabolism. Classical temperature models suggested that growth and various indices of nitrate metabolism all responded to temperature, with Q10 values of close to 2 over the whole temperature range. However, Q10 values over the interval from 8 to 17°C were higher than 2 and much lower than 2 between 17 and 25°C. Limitations to the Q10 concept are considered. Tem- perature effects on different measures of nitrate metabolism were very similar, supporting the hypothesis that the effects of temperature on diatom nitrate metabolism are mediated at the level of NR activity. Recent biochemical data for NR also supports this idea.

Diatoms are able to react to biotic and abiotic stress, such as competition, predation and unfavorable growth conditions, by producing bioactive compounds including polyunsaturated aldehydes (PUAs). PUAs have been shown to act against grazers and either enhance or inhibit the growth of different phytoplankton and bacteria both in culture and in the field. Presence of nanomolar concentrations of dissolved PUAs in seawater has been reported in the North Adriatic Sea (Mediterranean), suggesting that these compounds are released in seawater following diatom cell lysis. However, the origin of the PUAs and their effects on natural phytoplankton assemblages remain unclear. Here we present data from four oceanographic cruises that took place during diatom blooms in the northern Adriatic Sea where concentrations of particulate and dissolved PUAs were monitored along with phytoplankton cell lysis. Cell lysis was positively correlated with both concentrations of particulate and dissolved PUAs (R = 0.69 and R = 0.77, respectively), supporting the hypothesis that these compounds are released by cell lysis. However, the highest concentration of dissolved PUAs (2.53 nM) was measured when cell lysis was high (0.24 d(-1)) but no known PUA-producing diatoms were detected, suggesting either that other organisms can produce PUAs or that PUA-producing enzymes retain activity extracellularly after diatom cells have lysed. Although in situ concentrations of dissolved PUAs were one to three orders of magnitude lower than those typically used in laboratory culture experiments, we argue that concentrations produced in the field could induce similar effects to those observed in culture and therefore may help shape plankton community composition and function in the oceans.

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro- and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species (Apedinella spinifera), growth rate and final yield was no different between the media but in one case (Emiliania huxleyi) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species.

This paper describes inter-specific differences in the distribution of sediment in the gut compartments and in the enzyme and bacterial profiles along the gut of abyssal holothurian species — Oneirophanta mutabilis, Psychropotes longicauda and Pseudostichopus villosus sampled from a eutrophic site in the NE Atlantic at different times of the year. Proportions of sediments, relative to total gut contents, in the pharynx, oesophagus, anterior and posterior intestine differed significantly in all the inter-species comparisons, but not between inter-seasonal comparisons. Significant differences were also found between the relative proportions of sediments in both the rectum and cloaca of Psychropotes longicauda and Oneirophanta mutabilis. Nineteen enzymes were identified in either gut-tissue or gut-content samples of the holothurians studied. Concentrations of the enzymes in gut tissues and their contents were highly correlated. Greater concentrations of the enzymes were found in the gut tissues suggesting that they are the main source of the enzymes. The suites of enzymes recorded were broadly similar in each of the species sampled collected regardless of the time of the year, and they were similar to those described previously for shallow-water holothurians. Significant inter-specific differences in the gut tissue concentrations of some of the glycosidases suggest dietary differences. For example, Psychropotes longicauda and Pseudostichopus villosus contain higher levels of chitobiase than Oneirophanta mutabilis. There were no seasonal changes in bacterial activity profiles along the guts of O. mutabilis and Pseudostichopus villosus. In both these species bacterial activity and abundance declined between the pharynx/oesophagus and anterior intestine, but then increased along the gut and became greatest in the rectum/cloaca. Although the data sets were more limited for Psychropotes longicauda, bacterial activity increased from the anterior to the posterior intestine but then declined slightly to the rectum/cloaca. These changes in bacterial activity and densities probably reflect changes in the microbial environment along the guts of abyssal holothurians. Such changes suggest that there is potential for microbial breakdown of a broader range of substrates than could be otherwise be achieved by the holothurian itself. However, the present study found no evidence for sedimentary (microbial) sources of hydrolytic enzymes.

This overview compares and contrasts trends in the magnitude of the downward Particulate Organic Carbon (POC) flux with observations on the vertical profiles of biogeochemical parameters in the NE subarctic Pacific. Samples were collected at Ocean Station Papa (OSP, 50°N, 145°W), between 18–22 May 1996, on pelagic stocks/rate processes, biogenic particle fluxes (drifting sediment traps, 100–1000 m), and vertical profiles of biogeochemical parameters from MULVFS (Multiple Unit Large Volume Filtration System) pumps (0–1000 m). Evidence from thorium disequilibria, along with observations on the relative partitioning of particles between the 1–53 μm and >53 μm classes in the 50 m mixed layer, indicate that there was little particle aggregation within the mixed layer, in contrast to the 50–100 m depth stratum where particle aggregation predominated. Vertical profiles of thorium/uranium also provided evidence of particle decomposition occuring at depths ca. 150 m; heterotrophic bacteria and mesozooplankton were likely responsible for most of this POC utilisation. A water column carbon balance indicated that the POC lost from sinking particles was the predominant source of carbon for bacteria, but was insufficient to meet their demands over the upper 1000 m. While, the vertical gradients of most parameters were greatest just below the mixed layer, there was evidence of sub-surface increases in microbial viability/growth rates at depths of 200–600 m. The C:N ratios of particles intercepted by free-drifting and deep-moored traps increased only slightly with depth, suggesting rapid sedimentation even though this region is dominated by small cells/grazers, and the upper water column is characterised by long particle residence times (>15 d), a fast turnover of POC (2 d) and a low but constant downward POC flux.

The diel variation and regulation of the enzyme nitrate reductase (NR) were examined in the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle. NR was purified, and polyclonal antibodies were raised to a 98-kD polypeptide. The antibodies cross-reacted only with proteins from closely related diatom species, suggesting significant epitopic variation of this enzyme within algal divisions. Neither NR enzymatic activity nor protein was detected in cells grown with ammonium as the sole nitrogen source; the addition of ammonium to cells growing on nitrate decreased both protein levels and enzyme activity by 40% within 2 h. In cells grown on a 12:12 h LD cycle, NR activity and NR protein levels were highly correlated, with a peak at midday, a decrease toward the end of the photoperiod, and an increase in activity beginning near the end of the dark period. The addition of actinomycin D (an inhibitor of RNA synthesis) and cycloheximide (an inhibitor of protein synthesis) affected NR activity and NR protein levels identically, strongly suggesting that this nuclearencoded protein is regulated primarily at a transcriptional level. The diel pattern of NR protein and activity ceased immediately following transfer to continuous light, indicating that the periodicity is not directly controlled by a circadian rhythm. Time-lagged cross-correlation analysis revealed a 6-h phased difference between the minimum enzyme activity or protein levels and the maximum cellular carbon pool. On the basis of the experimental results, we develop a model proposing that (1) NR activity is regulated primarily by transcriptional regulation of NR synthesis and that (2) the level of expression of the enzyme during a given day is correlated with the integrated pool of organic carbon accumulated during the preceding photoperiod.